Summary/Abstract Homologousrecombinationisachromosomerepairprocessthatplaysessentialrolesinmeiosis,the specializedcelldivisionthatproducesgametes.Defectsinmeioticrecombinationarealeadingcauseof infertility,pregnancylossandcongenitaldiseaseinhumans.Amajorgapinourunderstandingofmeiotic recombinationisthemechanismoftherecombination-associatedDNAsynthesis(RADS)thatisessentialto restorechromosomeintegrity.Thisknowledgegappersistsbecauseofinherentchallengestostudyingmeiotic RADSinvivo,inparticulartheessentialnatureofDNAreplicationfactors,theneedtostudyRADSinisolation fromchromosomalreplication,andtherequirementforspecialassaystomeasureRADS.Thesehurdleshave nowbeenovercomeusinganinnovativecombinationofchemical,real-timeandmoleculargeneticstoolsin buddingyeastthatenableacuteinactivationofessentialreplicationfactorsspecificallyduringrecombination, andmeasurementofdenovoDNAsynthesis.ThissystemutilizesanATP-analogsensitivealleleoftheCdc7 kinase(cdc7-as3)tosynchronizecellsafterS-phase,butbeforerecombinationisinitiated.Real-time inactivationofessentialreplicationfactorsisachievedusingtheauxin-inducibledegron(AID)system,which hasbeenrewiredandoptimizedforuseinmeioticcells.TomonitorRADS,newlysynthesizedDNAislabeled, isolatedandquantifiedusing5-ethynyl-2?-deoxyuridine(Edu)incorporation,biotin-azideclickchemistry, streptavidinpurificationandquantitativePCR(qPCR).Exploitingthesetools,thelong-termobjectivesofthis projectaretounderstandthenature,function,mechanismandregulationofRADS.Theseobjectiveswillbe pursuedthroughthreeaims.Aim1willdeterminetheroleofRADSforboththeDNAeventsofmeiotic recombinationandthechromosomaleventsofmeioticprophaseusingthecomprehensivebatteryof molecular,geneticandcytologicalassaysuniquelyavailableinbuddingyeast.Aim2willtestmodelsofRADS bydelineatingthereplicationfactorsinvolvedandsystematicallyanalyzingtheirroles.Complementarystudies inmousewillanalyzethelocalizationanddynamicsofreplicationfactorsatsitesofrecombination.Aim3will identifyandcharacterizefactorsinvolvedintherecruitmentofreplicationfactorstorecombinationsitesandthe regulationofRADS.Immunofluorescencecytologywillbeusedtomonitorchromosomaldynamicsof replicationfactorsanddeterminethegeneticrequirementsfortheirlocalization.ThetimingandextentofRADS willbeanalyzedinstrainsmutantforfactorspredictedtomodulateRADS,includingmeiosis-specific recombinationproteins,DNAhelicasesandtopoisomerases.Theresultsoftheseaimswillprovide unprecedentedinsightsintothemechanismandregulationofRADS,fillingamajorgapinourunderstandingof meioticrecombination.Thesefindingswillbegermanetounderstandingpathologiesassociatedwithhuman meiosis,andareexpectedtodefineparadigmsthatarebroadlyrelevantforchromosomerepair.